During the reaction of an immune precipitate with fresh human serum, C3 undergoes a number of molecular alterations with the formation of conversion products differing from those obtained when purified components react. Those products which remain in the fluid phase, the subject of the present paper, have been identified by their reaction with monospecific antisera to the three antigenic determinants of C3, A, B, and D, after electrophoresis in agar or polyacrylamide gel.
When purified C3 reacts with EAC1,4,2, C3i is found in the fluid phase. C3i, a loose complex of C3a and C3b, is in a conformational state whereby only the A and D antigens, present on its C3b portion, will consume antibody. The B antigen, present on the C3a portion of C3i, is unavailable for combination with antibody until C3i dissociates.
In the fluid phase of the reaction of an immune precipitate with whole serum, C3i, C3a, and C3b, formed when purified components react, cannot be found. Instead the end products of the reaction appear to be C3c, which contains the A antigen, and C3d, which contains the D antigen. C3c and C3d are similar to the ß1A and α2D produced by the aging of serum but differ in their mobilities in acrylamide gel and in agar. The C3c and C3d generated by an immune precipitate also differ slightly from the C3c and C3d produced by the reaction of trypsin with C3 in whole human serum.
As human serum reacts with an immune complex, native C3 appears to undergo a primary alteration before conversion. This alteration results in a molecular species of C3 which is labile at 56°C for 30 min, fails to expose additional A and D antigenic sites upon aging, and which forms ß1A and C3d rather than ß1A and α2D during aging.
In addition to this altered form of native C3, a new conversion product, C3x, is formed as whole serum reacts with an immune complex. C3x is not found in systems utilizing pure complement components. C3x is like C3 in that it bears all three antigenic determinants but differs in that it has a slightly faster mobility in polyacrylamide gel than does native C3. C3x is not only found in the fluid phase but is also bound to the immune precipitate.
Finally, the fluid-phase kinetics of each of the antigens of C3 have been determined as normal human serum reacts with an immune precipitate. These illustrate that nearly the entire population of native C3 molecules undergoes conversion rapidly as manifested by the disappearance of the B antigen from the fluid phase. Moreover, the kinetics of the fluid-phase A and D antigens reflect that the conversion of C3 in serum is quantitatively not the same as when purified C3 reacts with C4,2.